Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter-loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter-loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter-loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.


Introduction
Cre-Lox recombination systems are versatile mouse genetics tools, permitting spatial-temporal control of gene expression [1][2][3][4][5][6] .Since their development in the 1980s 7,8 , they have become a widely accepted and extensively used method for genome editing, paving the way for new opportunities across various research elds.The applications of this increasingly potent technology are diverse, encompassing the generation of targeted deletions, translocations, or inversions, including double-oxed inverse orientation 9 .Cre-Lox applications also extend to trans-chromosomal recombination for generating deletions or duplications 10 , the Brainbow system 11,12 , which allows individual neurons to be marked by different colors, and to a growing array of inducible systems 13,14 .The advent of Cre-Lox technology has expedited the production of conditional knockout mice on a large scale 15,16 .Its utility is not limited to mice but extends to other research model animals such as rats, pigs, and zebra sh.The enduring relevance, interest, and continuing advancements of the Cre-Lox system are underscored by the high volume of publications using it.Since the beginning of 2024, 376 preprints related to 'Cre-Lox technology' have been shared on BioRxiv between January 1st and June 1st, 2024.This underscores the sustained interest and continuous advancements in the eld.
In its most basic form, the Cre-Lox system in mice consists of a Cre driver, which provides the Cre protein, a Cre reporter (e.g., lacZ or a uorescent reporter) and a oxed allele 8 .The oxed allele contains a target sequence-the region of Cre-mediated recombinationenclosed by a pair of directional loxP sites, each approximately 34 bp in length.The Cre protein binds the pair of loxP sites and initiates recombination between them 17,18 .The outcome of the recombination depends on the orientation of the loxP sites; recombination between loxP sites oriented in the opposite direction results in an inversion of the intervening sequence, while recombination between loxP sites oriented in the same direction results in deletion of the intervening sequence.Despite the proven value of the Cre-Lox technology, it comes with a hefty price tag and is time-consuming-the cost of generating conditional alleles is approximately $16,000 to $18,000, and the process takes around 7-9 months (Sr.Study Director, Genetic Engineering Technologies, The Jackson Laboratory, personal communication).Moreover, it is associated with speci c biological challenges [19][20][21][22][23][24] , including mosaicism of recombination within target tissues, which can lead to unexpected phenotypic outcomes (Fig. 1a).Even so, genetic mosaicism, a condition in which a multicellular organism possesses two or more populations of cells with different genetic makeup, is not necessarily an undesirable outcome for all applications.Mosaicism occurs in several human pathologies, including cancer, and therefore being able to predictably generate mosaic animals for translational research is as important as generating animals with a uniform genotype in every cell.8 litters, consisting of a total of 8 to 55 offspring, were then genotyped to assess the percent of three possible outcomes: complete recombination, mosaicism, or no recombination (Fig. 2b).
For the inter-loxP distance of 0.8 kb (Fig. 3a), the Ella-cre x Rosa-0.8 kb ox combination resulted in 54% of the offspring exhibiting recombination, while 33% displayed mosaic traits.The CMV-cre x Rosa-0.8 kb ox combination had a slightly higher recombination rate of 57%, with 43% showing mosaic patterns.Interestingly, 13% of the offspring in the Ella-cre x Rosa-0.8 kb ox combination retained the oxed state, a phenomenon absent in the CMV-cre x Rosa-0.8 kb ox combination.The Sox2-cre x Rosa-0.8 kb ox combination, however, demonstrated a higher recombination rate of 79%, with only 16% showing mosaic patterns and a mere 5% remaining oxed (Fig. 3b and Supplementary Fig. 1a).Further analysis was conducted to con rm that the mosaicism observed in tail DNA lysate from mosaic offspring was representative of the entire mouse.We employed probe-based assays to examine the following tissues: liver, spleen, kidney, skin, and brain.The patterns of recombination, or mosaicism, were compared between the tail DNA lysate and the tissue-speci c lysate.In most instances, we found a remarkable consistency in the patterns observed (Supplementary Fig. 1b).This consistency strongly suggests that tail DNA lysate could serve as a reliable predictor of the recombination/mosaic patterns in the entire organism.
Next, we examined the impact of Cre-driver strain on recombination and mosaicism at an inter-loxP distance of ~ 4 kb.The Ella-cre x Rosa-4 kb ox combination resulted in 16% of the offspring exhibiting recombination, while 60% displayed mosaic traits.The CMV-cre x Rosa-4 kb ox combination had a slightly higher recombination rate of 34%, with 53% showing mosaic patterns.Notably, 24% of the offspring in the Ella-cre x Rosa-4 kb ox combination retained the oxed state, compared to 13% in the CMV-cre x Rosa-4 kb ox combination.The Sox2-cre x Rosa-4 kb ox combination demonstrated a higher recombination rate of 73%, with only 27% showing mosaic patterns and 0% still oxed (Supplementary Fig. 2a).
We then investigated the impact of Cre-driver strain on Cre-mediated recombination and mosaicism at an inter-loxP distance of ~ 8 kb (Fig. 3c).The Ella-cre x Rosa-8 kb ox combination resulted in a mere 3% of the offspring exhibiting recombination, while 69% displayed mosaic traits.The CMV-cre x Rosa-8 kb ox combination had a slightly higher recombination rate of 14%, with 72% showing mosaic patterns.Interestingly, 28% of the offspring in the Ella-cre x Rosa-8 kb ox combination retained the oxed state, compared to only 14% in the CMV-cre x Rosa-8 kb ox combination.The Sox2-cre x Rosa-8 kb ox combination demonstrated a higher recombination rate of 37%, with only 34% showing mosaic patterns and 29% remaining oxed (Fig. 3d).
Building upon these observations, our exploration delved further into understanding the nuances of genetic in uence.Particularly intriguing were the ndings when examining the tumor suppressor Trp53 locus (Supplementary Fig. 2b).When we crossed Ella-cre with Trp53-8 kb ox, we observed that 19% of the offspring exhibited recombination.A higher recombination rate of 72% was noted when the CMV-cre strain was crossed with the Trp53-8 kb ox.However, the offspring of Sox2-cre and Trp53-8 kb ox demonstrated a higher recombination rate of 82%.In comparison to the Rosa-8 kb ox F1 offspring (Fig. 3d), the offspring of Trp53-8 kb ox showed higher recombination rates with all three Cre drivers (Supplementary Fig. 2b).This suggests that genetic loci can also impact Cremediated recombination and mosaicism.
Genes can circumvent knockout strategies and reinitiate transcription through various mechanisms such as exon skipping, nonsenseassociated altered splicing, or the use of cryptic splice sites [45][46][47][48] .This highlights the importance of knocking out the entire gene rather than just the critical exons to prevent any unexpected mRNA transcripts 45,46 .Our investigation into the lengths of all proteincoding genes in mice (Supplemental Table 1; The data was sourced from Mouse Genome Informatics 49 ) and the subsequent nding that the median length is 15.8 kb provided a valuable reference point (Fig. 3e).For a comprehensive examination of Cre-recombinase e ciency at inter-loxP distances of up to 15 kb, we designed three new strains at the ROSA26 locus-Rosa-10 kb, Rosa-12 kb, and Rosa-15 kb.Because the recombination e ciency of the Sox2-cre strain was superior (Figs.3b and 3d), we used this strain to investigate recombination e ciency at these three new inter-loxP distances.The data suggest that beyond an inter-loxP distance of 10 kb, recombination without mosaicism becomes less likely.This is evident from the Sox2-cre x Rosa-10 kb ox cross resulting in only 22% of offspring exhibiting recombination (Fig. 3f), and the Sox2-cre x Rosa-12 kb (Fig. 3g) and Rosa-15 kb (Fig. 3h) ox combinations showing no recombination, but 14% mosaicism at an inter-loxP distance of 12 kb.
Collectively, these ndings provide valuable insights into the dynamics of Cre-mediated recombination and mosaicism relating to Cre driver strain.They underscore the superior recombination e ciency of the Sox2-cre strain at inter-loxP distances of up to 10 kb, while concurrently highlighting the in uence of the genetic locus.We observed a notable decrease in recombination e ciency correlating with an increase in inter-loxP distance across all three Cre-driver strains.Considering these ndings, we propose that researchers design oxed alleles with an inter-loxP distance of less than 4 kb to achieve complete recombination, and greater than 8 to 10 kb for mosaic recombination.This recommendation is based on the observation that over 73% of offspring from the Sox2-cre x Rosa-4 kb cross exhibited completely recombined alleles, and greater than 72% of offspring from the CMV-cre x Rosa-8 kb cross exhibited mosaicism.To further enhance recombination e ciency and mosaicism in F1 crosses, we advocate utilizing the Sox2-cre and either the Ella-cre or CMV-cre driver strains, respectively.
Inter-loxP distance of 3 kb or less is optimal for e cient recombination when using mutant loxP sites.
We next examined how changes in the distances between loxP sites in mutant loxP oxed alleles affected recombination e ciency and mosaicism.This was accomplished using ve unique double-oxed inverted open reading frame (DIO) mouse strains, which contain wildtype loxP and mutant loxP (lox2272) each with different inter-loxP distances-1.1,2.2, 5.3, 6.9, and 8.1 kb (Supplementary Fig. 3a).One of the signi cant challenges, especially when creating oxed DIO alleles with different inter-loxP distances at a xed locus, has been the lack of a precise method for integrating DIO transgenes ranging from 1 kb to 8 kb.The currently available CRISPR/Cas9 technology is slow and presents di culties due to the variability and ine ciency of CRISPR/Cas9-mediated homologydirected repair for inserting transgenes larger than 1-3 kb.Furthermore, the insertion of loxP sites using CRISPR/Cas9 introduces its own set of challenges 16 -the e ciency of generating conditional knockout alleles by simultaneously inserting right and left loxP sites via the delivery of 2 single guide RNAs (sgRNAs) and 2 single-stranded oligo DNA nucleotides (ssODNs) as donors is less than 1%.To address these issues, we utilized a high-e ciency Bxb1 recombinase system that we recently developed 40 to individually integrate ve constructs with different inter-loxP site lengths into the ROSA26 locus (Fig. 4a).This approach allowed us to rapidly, e ciently, and uniformly generate ve new DIO oxed strains.
Initially, we examined the impact of the Ella-cre driver strain on recombination and mosaicism across ve mutant inter-loxP distances, ranging in length from 1.1 kb to 8.1 kb (Fig. 4b).For the offspring of the Ella-cre x Rosa-DIO-1.1 kb, an inter-loxP distance of 1.1 kb resulted in a mere 8% of the offspring showing recombination, while a noteworthy 88% displayed mosaic characteristics, and 4% remained oxed.Notably, the Ella-cre x Rosa-DIO-2.2 kb cross led to a marginally better recombination rate of 16%, with 59% demonstrating mosaicism, and 23% remaining oxed.In the scenario of the Ella-cre x Rosa-DIO-5.3 kb cross, none of the offspring exhibited complete recombination, while 65% showed mosaicism, and 34% stayed oxed.For the Ella-cre x Rosa-DIO-6.9 kb pairing, 4% of the offspring showed full recombination, while 37% displayed mosaicism, and a 58% stayed oxed.Finally, in the Ella-cre x Rosa-DIO-8.1 kb DIO cross, 3% of the offspring showed full recombination, while a majority of 68% displayed mosaicism, and 28% stayed oxed (Fig. 4b).
Next, we delved into the impact of the CMV-cre-driver strain on recombination and mosaicism across ve diverse mutant inter-loxP distances (Fig. 4c).With the Rosa-DIO-1.1 kb allele, the CMV-cre F1 progeny yielded a recombination rate of 38%, exhibited mosaicism in 57% of instances, and roughly 4% remained oxed.In the scenario of the Rosa-DIO-2.2 kb allele, 33% of the progeny demonstrated full recombination, 52% manifested mosaicism, and 13% remained oxed.In the case of the Rosa-DIO-5.3 kb allele, 14% of the progeny showed full recombination, a notable 71% displayed the mosaic state, and 14% remained oxed.For the Rosa-DIO-6.9 kb allele, 13% of the progeny exhibited full recombination, 65% displayed the mosaic state, and 21% remained oxed.Finally, for the Rosa-DIO-8 kb allele, 14% of the progeny demonstrated full recombination, a majority of 71% preserved the mosaic state, and 14% remained oxed (Fig. 4c).Lastly, we investigated the impact of the Sox2-cre strain on recombination and mosaicism across ve mutant inter-loxP distances (Fig. 4d).Upon analyzing the Rosa-DIO-1.1 kb allele, we observed that 81% of the Sox2-cre F1 progeny demonstrated recombination, 16% displayed mosaicism, and a small fraction of 2% remained without any recombination.When considering the Rosa-DIO-2.25 kb allele, we found that 71% of the progeny showed complete recombination, while 14% presented with the mosaic state, and an equivalent percentage remained oxed.In the case of the Rosa-DIO-5.3 kb allele, 53% of the progeny exhibited full recombination, 34% displayed the mosaic state, and 11% were oxed.Regarding the Rosa-DIO-6.9 kb allele, 41% of the progeny showed complete recombination, 33% showed a mosaic state, and 25% remained oxed.Finally, for the Rosa-DIO-8.1 kb allele, a mere 18% of the progeny demonstrated complete recombination, 47% were mosaic, and 34% remained oxed.
To investigate Cre-Lox mosaicism at the single-cell level, we used a cross with a high proportion of mosaic F1 progeny, CMV-cre x Rosa-DIO-8.1 kb.Upon recombination, the Rosa-DIO-8.1 kb allele induced the expression of the inner ear sensory cell-speci c transcript Myo15a conjugated to the self-labeling HaloTag 50 .The neatly patterned single layer of cells in the cochlear sensory epithelium provides an ideal canvas for viewing cell-by-cell mosaicism.We isolated cochleae from F1 pups at postnatal day 2, labeled them with TMR-conjugated HaloTag ligand, and imaged the sensory epithelium.The mosaic pattern was striking, showing a clear variegation of sensory cells with and without HaloTag labeling at the distal cytoskeletal projections in a single eld of view (Fig. 4e).Four different F1 mosaic offspring were analyzed, and the degree of mosaicism varied by individual, ranging from 37-76% of cells recombined.On average, a slight majority of cells were recombined, at 55% (Fig. 4f).Notably, tail-tip PCR-based genotyping of the CMV-cre x Rosa-DIO-8.1 kb F1 offspring (28 offspring from 4 litters) showed 71% mosaicism (Fig. 4c).This highlights the potential challenge of Cre-Lox con gurations prone to mosaicism: experimental outcomes may be skewed due to the presence of unexpected mosaic individuals.Conversely, there may be cases in which having both recombined and still-oxed "escaper" cells within a single eld of view can be advantageous, as cells that fail to recombine could act as internal controls for phenotypic comparisons of neighboring cells (manuscript under review).
Our experimental ndings collectively indicate that an increase in the DIO inter-loxP distance from 1.1 to 8.1 kb is associated with a corresponding decrease in the percentage of recombined loci.It is noteworthy that the offspring of the Ella-cre x DIO cross exhibited the lowest recombination levels.Speci cally, even with the 1.1 kb DIO allele, the percentage of recombined loci was less than 20%, and this percentage was even lower for all other tested Ella-cre x DIO progeny.Interestingly, no recombination was observed in the offspring of the Ella-cre x DIO-5.3 kb cross.However, among the three Cre-driver strains tested, Sox2-cre demonstrated superior recombination e ciency; the Sox2-cre x DIO-1.1 kb and Sox2-cre x DIO-2.2 kb crosses surpassed a recombination rate of 70%.The next tested distance (DIO-5.3kb inter-loxP distance) resulted in a recombination rate of approximately 50%, and the percentage of mosaic loci steadily increased from 14% for DIO-2.2 kb to 34% for DIO-5.3 kb.This trend persisted when the mutant inter-loxP distance was extended from 5.3 kb to either 6.9 kb or 8.1 kb.These results underscore the complex relationship between the inter-loxP distance, mutant loxP sites, recombination e ciency, and the speci c Cre-driver strain used in the study.Based on these observations, we suggest that researchers design DIO oxed alleles with an inter-loxP distance of less than 3 kb and ~ 8 kb to maximize recombination e ciency versus mosaicism, respectively.This suggestion is based on the nding that over 71% of offspring from the Sox2-cre x DIO-2.2 kb cross exhibited completely recombined alleles and approximately half (47%) or more of offspring from all Cre x DIO-8.1 kb cross exhibited mosaicism.
Comparative analysis of wildtype and mutant loxP sites: impact on recombination e ciency and mosaicism.
To more precisely evaluate the impact of using wildtype versus mutant loxP sites on recombination e ciency and mosaicism, we generated two distinct mouse strains, both with a xed inter-loxP distance of 6.9 kb, but at different loci (Rhbdf2 and Rhbdf1), with different loxP sites, either wildtype or mutant lox71/66 sites (Fig. 5a).We selected these loci because of our prior experience with them as well as their relevance to human disease (see below).Our ndings indicate that the use of wildtype loxP sites considerably improves recombination e ciency.This was evident when we compared the recombination e ciency of wildtype loxP (6.9 kb) and lox71/66 (6.9 kb) using three different Cre-driver strains.The complete recombination e ciency improved from 4% with Ella-cre, to 13% with CMV-cre, and 42% with Sox2-cre strain when utilizing wildtype loxP sites.However, it is important to note that none of the three Cre-driver strains induced complete recombination in the mouse strain with mutant loxP sites (Fig. 5a).In a similar vein, when we compared wildtype loxP at the Rhbdf2 locus (2.7 kb) and lox71/66 at the Rhbdf1 locus (2.9 kb) using the Sox2-cre strain, we observed a similar trend (Fig. 5a).There was an improvement in recombination e ciency from 42-72% when the inter-loxP distance was reduced from 6.9 to 2.7 kb while utilizing the wildtype loxP sites (Fig. 5a-b).Interestingly, we observed a lower recombination rate increase in Sox2-cre x 2.9 kb Mut lox71/66 F1 progeny, reduced by 22 percentage points when compared to the Sox2-cre x 2.7 kb WT loxP F1 progeny.However, there was an increase from 0-50% in complete recombination rate in Sox2-cre x 2.9 kb Mut lox71/66 F1 progeny compared with the Sox2-cre x 6.9 kb Mut lox71/66 F1 progeny (Fig. 5c).These data suggest that when the inter-loxP distance was reduced from 6.9 kb to 2.9 kb, even when utilizing the mutant loxP sites, there was clear improvement in recombination.
To deepen our understanding of our research ndings, we performed a thorough analysis of the distance between loxP sites using mutant lox71/66 oxed alleles.The Rhbdf1 gene, a member of the rhomboid family, has been associated with human cancer 51,52 .In previous studies, we engineered mice to be constitutively homozygous-null for Rhbdf1 (Rhbdf1 -/-) 45 .These mice displayed growth retardation, brain hemorrhage, cardiac hypertrophy, and unfortunately, did not survive beyond the 14th postnatal day 45 .To discern the speci c role of Rhbdf1 in various tissues, we engineered Rhbdf1 conditional-ready mice (Rhbdf1 ox/ ox-6.9kb ).This was accomplished by positioning the lox71 site in intron 1-2 and the lox66 site in exon 18, which are approximately 6.9 kb apart (Supplementary Fig. 4).Both mutant loxP sites were con rmed to be correctly targeted, with no unintended mutations, through PCR and Sanger sequencing.
We rst bred these Rhbdf1 ox/ ox-6.9kbmice with Ella-cre, CMV-cre, and Sox2-cre mice to produce tissue-wide homozygous-null mice.We anticipated that the deletion of Rhbdf1 across all tissues would lead to multiple organ pathologies.However, the offspring from these crosses, where breeders were hemizygous for Sox2-cre (or homozygous for either Ella-or CMV-cre) and homozygous for the Rhbdf1 oxed allele, were unexpectedly healthy, with no observable phenotypic or histological abnormalities (Supplementary Fig. 5).Further analysis utilizing PCR and probe-based assays revealed that despite the expression of the Cre transgene in all tested tissues, the recombinase failed to induce excision in most tissues.This resulted in mosaic recombination, leading to a viable phenotype.We hypothesized that the inter-loxP distance of 6.9 kb with the mutant loxP sites might be too large for e cient recombination at the Rhbdf1 locus.To test this hypothesis, we used CRISPR/Cas9-mediated gene editing to reduce the inter-loxP distance from 6.9 kb to 2.9 kb.This was achieved by deleting non-critical exons 4 through 12, thereby creating Rhbdf1 ox/ ox-2.9kbmice (Supplementary Fig. 4).We have previously demonstrated that the deletion of these non-critical exons does not affect the viability or fertility of mice 45 .
Finally, we crossed these Rhbdf1 ox/ ox-2.9kbmice with Sox2-cre driver strain mice.This resulted in the deletion of exons 2 through 18 via complete recombination and the expected phenotype-growth retardation, cardiac hypertrophy, and postnatal lethality (Supplementary Fig. 5b-d).Importantly, it appears that the Rhbdf1 locus does not have stoichiometric limitations on Cre activity.This is based on our observation of ine cient Cre-recombination at other loci (8.1-kb DIO oxed-Myo15a allele with CMV-cre; Fig. 4e).Collectively, these ndings suggest that 7 kb exceeds the allowable length for effective Cre-mediated recombination using these Cre driver strains at this locus.
The knockout of both Rhbdf1 and Rhbdf2 genes results in embryonic lethality 45 .Interestingly, when we attempted to replicate this phenotype using a combination of Rhbdf1 ox/ ox-6.9kballele, Sox2-cre, and Rhbdf2 -/-mice, we were unable to do so (Fig. 5d, upper panel).However, when we used the Rhbdf1 ox/ ox-2.9kballele in combination with Sox2-cre and Rhbdf2 -/-mice, the offspring exhibited embryonic lethality (Fig. 5d, lower panel).This nding supports our observation that for e cient Cre-recombination at the Rhbdf1 locus, the optimal distance between mutant loxP sites is less than 3 kb.Furthermore, we discovered that the 'open eyelid birth' phenotype in Rhbdf1 and Rhbdf2 double knockout mice is regulated by the keratinocyte-speci c expression of Rhbdf1 and Rhbdf2 (Fig. 5e, lower panel), as it is reproducible using a tissue-speci c K14-cre driver.Again, lack of complete recombination in Rhbdf1 ox/ ox-6.9kb, K14-cre, Rhbdf2 -/-mice, failed to show the 'open eyelid birth' phenotype with keratinocyte-speci c knockout (Fig. 5e, upper panel).We also found that endothelial-speci c expression is crucial for survival.This was evidenced by the observation that the endothelial-speci c deletion of Rhbdf1 in Rhbdf2 knockout mice using the Cdh5-cre driver resulted in embryonic lethality (Fig. 5f).These ndings provide valuable insights into the role of Rhbdf1 and Rhbdf2 in embryonic development and survival.
Overall, these ndings shed light on the consequences of Rhbdf1-KO mosaicism, emphasizing the importance of considering inter-loxP distance in genetic studies, especially in the presence of mutant alleles.
Impact of zygosity on recombination e ciency in Cre-Lox system.
We next explored the impact of zygosity of the oxed alleles on recombination e ciency (Fig. 6a).We created a series of breeding schemes using Ella-cre (homozygous) or Sox2-cre (hemizygous) females crossed with either heterozygous or homozygous males from a variety of oxed strains.Strains tested included both wild-type and mutant loxP sites, both ROSA26 and Rhbdf1 genetic loci, and a range of inter-loxP distances from 0.8 kb to 8 kb.For heterozygous crosses, only offspring that inherited a parental oxed allele, regardless of recombination state, were considered.In the case of Ella-cre x Rosa-2.25 kb, we found that heterozygous offspring displayed 54% mosaicism and 38% recombination, while homozygous offspring exhibited 50% mosaicism and only 14% recombination (Fig. 6b).These results indicate that heterozygosity led to higher complete recombination e ciency in this breeding scheme.Our examination of other oxed alleles and the Sox2-cre driver strain yielded consistent results.
In all breeding schemes, regardless of Cre driver strain, wildtype or mutant loxP, genomic locus or inter-loxP distance, heterozygous male breeders for the oxed allele yielded better recombination e ciency compared to homozygous male breeders.Collectively, these ndings highlight the relationship between zygosity and recombination e ciency/mosaicism in our experimental setup.
Impact of breeder age on recombination e ciency in Cre-Lox experiments.
The age of Cre-driver strain breeders is another variable with potential impacts on recombination e ciency.To investigate this, we used female CMV-cre and Sox2-cre mice and collected litters from breeders ranging in age from 5 to 38 weeks old at the time of mating (Fig. 7a).Furthermore, we tested four different oxed alleles with inter-loxP distances ranging from 0.8 kb to 8 kb.We determined the percentage of recombined pups from a total of 4-8 litters for each oxed strain.
Recombination e ciency varied between litters for all four oxed alleles.In general, it was lower in litters derived from very young Cre breeders (less than 10 weeks of age at the time of birth) and older breeders (more than 25 weeks).To determine whether breeder age was truly correlated with recombination rate, we tted a non-linear curve to each oxed allele data set.The correlation was strongest (R² = 0.998) for the Sox2-cre x Trp53-8 kb allele, with a slightly positive, nearly linear relationship between Cre breeder age and the percentage of recombined progeny.However, this trend was an exception, as the other three alleles each t best to a downwardopening parabolic curve (R² = 0.759, 0.333, and 0.623, respectively for CMV-cre x Rosa-2.25 kb, Sox2-cre x Rosa-6.9kb, and Sox2-cre x Rosa-0.8kb), indicating an ideal breeder age in the median of the range, with a consistent consensus of approximately 20 weeks yielding peak e ciency.For three of the four oxed strains tested, litters collected from Cre breeders older than 20 weeks faced a decline in recombination e ciency.This decline was not observed in the Sox2-cre x Trp53-8 kb litters, which may be attributed to a lack of data points for advanced age (more than 20 weeks) Cre breeders compared to the other oxed strains measured (Fig. 7b).
We therefore conclude that the age of the Cre-driver strain at the time of breeding should be an additional consideration in Cre-Lox experimental design.This data corroborates previous, limited evidence supporting age-based effects in Cre-driver mouse strains 53 .For optimal recombination rate, we recommend using Cre breeders that are at least 8 weeks old, but no older than 20 weeks.E ciency of Cre mediated recombination across different genomic loci in conditional knockout mouse strains.
A majority of our studies herein focused on Rosa oxed alleles, which have the bene t of consistency with respect to genomic loci.However, studies involving Cre-Lox for the purpose of conditional knockout, for example, will require Cre recombination targeted to other regions of the genome.Genomic locus should therefore be considered as an additional variable affecting recombination rate.
To address this question, we used the Sox2-cre strain crossed with twelve different conditional knockout mouse strains with oxed alleles across different regions of the mouse genome (Fig. 8 and Supplementary Figs.6-13).This panel spanned a total of nine autosomal chromosomes.All conditional knockout alleles had inter-loxP distances < 4 kb to support optimal recombination, with a range of 0.8 kb to 3 kb.
Irrespective of chromosomal location, recombination e ciency was high for each cross, ranging from 85-100% recombined F1 offspring.Rates of mosaicism in offspring were low, ranging from 0-11% of offspring, and failure to recombine was rare, ranging from 0-7% still oxed.Five of the twelve crosses showed 100% recombination, with oxed alleles on chromosomes 1, 6, 11, 15, and 19.
Mosaicism was observed in offspring from crosses with oxed alleles on chromosomes 1, 4, 5, 7, and 8.Only two crosses yielded litters with any non-recombined progeny: Sox2-cre x Chr.11 2 kb ox and Sox2-cre x Chr.5 0.8 kb ox.Thus, chromosome location does not appear to be a strong predictor of recombination e ciency in this study.It is informative to compare the crosses with oxed alleles on the same chromosome, for example Sox2-cre x Chr.11 2 kb ox (93% recombined) vs. Chr.112.7 kb ox (100% recombined), or Chr.5 0.8 kb ox (85% recombined) vs. Chr.5 3 kb ox (93% recombined).These cases demonstrate that the e ciency of recombination can indeed vary.This variation is minor, however, and is based on the genomic locus, even when considering loci on the same chromosome (Fig. 8).
Therefore, we conclude from this panel that conditional knockout alleles with an inter-loxP distance < 4 kb should have high recombination e ciency regardless of the oxed allele's location in the mouse genome.

Discussion
The exibility of the Cre-Lox system extends beyond its standalone capabilities.It can be seamlessly integrated with other geneediting systems, such as CRISPR-Cas9 and Cas12a, thereby expanding its potential applications and empowering researchers with a toolkit for genetic manipulation.Over the years, the Cre-Lox system has been employed in various groundbreaking studies.For instance, it played a crucial role in the development of techniques for identifying speci c cell types, such as Brainbow, which enables the labeling and distinction of individual neurons, advancing our understanding of complex neural networks 12,29 .Notably, it was instrumental in generating a mouse strain for studying COVID-19 54 and played a role in the development of vaccines for avian in uenza 55 .
Despite the utility of the Cre-Lox system for genetic engineering, the use of Cre is associated with several limitations.First, Cre expression can be unpredictable and "leaky," potentially interfering with the intended results.For example, the unexpected ectopic expression of Cre can lead to unanticipated recombination events during development.Second, inducible Cre expression may vary across different tissue types, with common inducers, like tamoxifen, exhibiting tissue-speci c variations.Third, high levels of Cre expression have been associated with toxicity, although tamoxifen has also been implicated as a potential factor in the case of conditional CreER T drivers.In addition, e cient use of Cre-Lox is hindered by factors not speci c to Cre expression.For example, not all tissue-speci c promoters are truly speci c, which can result in gene expression in unintended cell populations.In addition, differences among genes in their propensity for recombination may be in uenced by the physical structure of chromatin.These previously documented issues collectively contribute to unexpected phenotypic outcomes, posing a signi cant obstacle to the generation of faithful mouse models of human diseases.
The ndings we present here provide guidance to help researchers use Cre-Lox technology as effectively as possible in the face of the above limitations.With respect to the choice of a cre-driver strain, we show that Sox2-cre is more effective than Ella-cre or CMV-cre in achieving recombination.Furthermore, by examining varying inter-loxP distances (0.8 kb, 4 kb, 8 kb, 10 kb, 12 kb, and 15 kb), our work sheds light on how these distances impact recombination rates and mosaicism, aiding in the design of future experiments involving Cre-mediated recombination.Moreover, our ndings suggest that tail DNA lysate could serve as a reliable predictor of recombination/mosaic patterns in the entire organism, streamlining the process of analyzing recombination and mosaicism in genetic studies.
Our results show that an inter-loxP distance of less than 3 kb is optimal for recombination when utilizing mutant loxP sites.To investigate the effects of mutant loxP sites on recombination e ciency, with a special emphasis on the role of varying inter-loxP distances in mutant loxP oxed alleles, we employed a high-e ciency Bxb1 recombinase system to incorporate ve constructs, each with different inter-loxP site lengths, into the Rosa26 locus.This strategy expedited the creation of ve novel double-oxed inverted open reading frame (DIO) mouse strains.Our observations indicate that recombination e ciency diminishes as the inter-loxP distance extends from 1 kb to 8 kb.This insight is pivotal for the design of genetic experiments that involve Cre-mediated recombination.Our research also unveils that different Cre-driver strains, namely Ella-cre, CMV-cre, and Sox2-cre, have diverse impacts on recombination and mosaicism.For example, the progeny from the Ella-cre x DIO crosses demonstrated the lowest levels of recombination, with less than 20% recombination even with the 1.1 kb DIO allele.In contrast, crosses involving the Sox2-cre driver strain exceeded a recombination rate of 70% with inter-loxP distances of 1 kb and 2.25 kb.This suggests that DIO oxed alleles with inter-loxP distances of less than 3 kb enable e cient recombination.Moreover, the research uncovers intriguing patterns in mosaicism.As the inter-loxP distance expands, the percentage of mosaic individuals also escalates, implying that larger inter-loxP distances may result in higher incidences of mosaicism.This could be a signi cant factor to consider when designing oxed alleles with mutant loxP sites.
Our research also explored the relationship between the age of Cre-driver strain breeders and the degree of mosaicism in their offspring, revealing that breeders aged between 10 and 20 weeks were most likely to achieve successful recombination.We embarked on this investigation by employing CMV-cre and Sox2-cre strains, collecting litters from breeders whose ages ranged from 5 to 38 weeks at the time of mating.We tested four distinct oxed alleles, with inter-lox distances spanning from 0.8 kb to 8 kb.We observed that recombination e ciency varied across litters for all four oxed alleles, with a general trend of lower e ciency in litters from extremely young breeders (under 10 weeks) and older breeders (over 25 weeks).Notably, for three out of the four oxed strains tested, litters from Cre breeders older than 20 weeks exhibited a decline in recombination e ciency.These ndings underscore the importance of considering the age of the Cre-driver strain at the time of breeding when designing Cre-Lox experiments.This may be a concern in experimental designs that use multiple litters from the same breeding pair over time as replicates, where temporal changes in recombination e ciency may produce variable results.
We also investigated the in uence of zygosity on the e ciency of recombination in oxed alleles.A series of breeding schemes was established, involving both homozygous and heterozygous crosses with a variety of oxed strains.The strains tested encompassed both wildtype and mutant loxP sites, different genetic loci (ROSA26 and Rhbdf1), and a range of inter-loxP distances.Our ndings indicate that heterozygosity consistently led to higher recombination e ciency across all tested parameters.This was observed even in less e cient crosses, suggesting a robust relationship between zygosity and recombination e ciency.For best reproducibility, parental zygosity should be consistent in experiments using multiple litters.
We examined the impact of using wildtype versus mutant loxP sites on recombination e ciency and mosaicism in mouse models.In particular, our ndings from two distinct mouse strains, one with wildtype loxP sites and the other with mutant lox71/lox66 sites, suggest that the use of wildtype loxP sites improves recombination e ciency.We found that when comparing the e ciency of wildtype loxP and lox71/lox66, the recombination e ciency for a 6.9 kb inter-loxP allele improved from 0-4%, 13%, or 42% depending on the Cre-driver strain.Further analysis was performed to understand the impact of inter-loxP distance on recombination e ciency.
By reducing the inter-loxP distance from 6.9 kb to 2.9 kb, even when utilizing mutant loxP sites, there was a dramatic increase in recombination, rising from no recombination to 50%.We also explored the speci c role of the Rhbdf1 gene in various tissues.Rhbdf1 conditional-ready mice were generated by positioning lox71 and lox66 sites approximately 6.9 kb apart.Surprisingly, homozygous-null mice resulting from crosses with Cre-driver strains did not display expected phenotypic abnormalities, indicating ine cient recombination.To address this, the inter-loxP distance was reduced to 2.9 kb using CRISPR/Cas9-mediated gene editing.This modi cation resulted in complete recombination and the expected phenotype in offspring.Crucially, our ndings suggest that for e cient Cre-recombination, the optimal distance between mutant loxP sites is less than 3 kb.This conclusion was supported by experiments attempting to replicate the embryonic lethality phenotype seen with the knockout of both Rhbdf1 and Rhbdf2 genes.Overall, our ndings underscore the importance of considering inter-loxP distance in genetic studies, especially when dealing with mutant oxed alleles.
Our research, while primarily centered on an array of ROSA26 oxed alleles, also encompasses a broader genomic scope.In this context, we employed the Sox2-cre strain in conjunction with twelve distinct conditional knockout mouse strains.These strains possessed oxed alleles with inter-loxP distances of < 4 kb, scattered across various regions of the mouse genome.The outcomes were promising, with all crosses demonstrating a high recombination e ciency, ranging from 85-100%, regardless of their chromosomal positions.Furthermore, the instances of mosaicism in offspring were minimal, and instances where recombination failed to occur were exceedingly rare.These ndings lead us to propose that, irrespective of their genomic location, conditional knockout alleles with an inter-loxP distance of < 4 kb are likely to exhibit high recombination e ciency.This insight holds signi cant implications for genetic manipulation in mouse models, and is encouraging for studies involving conditional knockout, for example, where the locus of the oxed allele cannot be controlled.
In conclusion, our study systematically optimized Cre-mediated recombination and e ciency in mice and provides guidelines for e cient design of oxed alleles with wildtype and mutant loxP sites.Additionally, we provide guidelines for optimal inter-loxP distance for e cient recombination.Our ndings underscore the complex interplay of factors such as inter-loxP distance, Cre driver strain, breeder age, and zygosity on recombination e ciency.This highlights the necessity of ne-tuning experimental conditions to yield robust and reproducible results.Key ndings of our research include the exceptional performance of the Sox2-cre strain and an optimal inter-loxP distance of less than 4 kb when using wildtype loxP sites for recombination.We discovered that a reduction in the inter-loxP distance from 6.9 kb to 2.9 kb enhanced recombination, even when using mutant loxP sites.This suggests that for e cient Cre-recombination, the optimal distance between mutant loxP sites is less than 3 kb.Expanding beyond ROSA26 oxed alleles, our study explores a wider genomic landscape, demonstrating that high recombination e ciency can be achieved across diverse genomic locations, provided the inter-loxP distance is < 4 kb.These collective ndings deepen our understanding of Cre-mediated recombination, providing a solid foundation for designing future genetic experiments.
Our research opens up new avenues for generating mosaic animals characterized by sporadic gene activation.Producing a mosaic of recombined and still-oxed "escaper" cells can be very convenient for analysis of cellular features at the single-cell or tissue level.For example, we recently used non-recombined sensory cells in the cochlea as internal controls for neighboring conditional knockout cells while measuring the length of cytoskeletal projections (manuscript under review).This ensured that our comparisons were as accurate as possible, given that cellular features change depending on cochlear position.Measurements comparing directly adjacent cells are only possible with mosaic recombination.
Mosaic recombination also has a unique advantage for cancer research.It is worth noting that there is a fundamental difference between a transgenic model, where an oncoprotein is expressed in every cell of a tissue or cell lineage, and human cancers, where this is not the case.These mosaic animals could prove to be invaluable tools for simulating human cancer, particularly when germline embryonic transgene expression results in lethality 56 .For example, the progeny of K-ras oxed mice and Protamine-cre mice (which is active in haploid sperm) exhibit embryonic lethality due to the expression of the K-Ras oncoprotein during early development 57 .To overcome this issue and establish a tumorigenesis model using sporadic K-ras activation (instead of widespread activation), Guerra et al. produced offspring by crossing K-ras oxed mice with CMV-cre mice 58 .This resulted in a signi cant number of offspring reaching adulthood, presumably due to incomplete CMV-cre mediated recombination of the K-ras oxed allele.Furthermore, signs of pathology, such as respiratory problems, were observed when the progeny reached seven to eight months old 58 , suggesting that mosaicism can reduce the tumor burden.Our systematic analysis of different inter-loxP distances, different types of loxP sites, the age of the breeder strains, and the zygosity of the oxed alleles, should facilitate the creation of better mouse models of cancer.
Notably, as reported by Guerra et al., reduction in tumor burden due to mosaicism could potentially prolong the lifespan of the mice, enabling longer-term studies.The knowledge gained here, along with alterations in the number of adeno-associated virus particles delivered, an increase in the wide availability of AAV-cre, and the combination of AAV serotype and promoter speci city, can help to selectively activate or silence conditional alleles, thereby generating non-germline mouse models to enable the study of cancer at single-cell resolution.

Zygote electroporation or microinjection
The process of gene editing was performed using two distinct methods: microinjection (large DNA transgenesis using Bxb1 mRNA) or electroporation (CRISPR/Cas9-mediated gene deletion).The procedures for both electroporation and microinjection were carried out according to previously published protocols 40,42,59 .Brie y, zygotes were harvested from C57BL/6J females that had undergone superovulation and mating.These zygotes were then placed in a specially prepared embryo collection medium.Following this, the zygotes were either immediately subjected to manipulation or kept in an incubator at a temperature of 37°C with 5% CO2 until the time of manipulation and transfer.After the manipulation process, the embryos were transferred into pseudopregnant recipients.

Genotyping
Pregnant dams were euthanized by CO 2 asphyxiation, performed in a manner consistent with the 2013 recommendations of the American Veterinary Medical Association Guidelines on Euthanasia.Offspring from Cre-driver x oxed strains were collected from pregnant mice at E17.5.Pups P1 to P3 were decapitated.Tail tips, no longer than 2 mm, were excised for DNA preparation.Tail tips were either stored overnight at 4°C or directly placed in 100 µl of lysis buffer containing 1.5 µl of Proteinase K, then incubated overnight at 55°C.Following this, they were denatured at 95°C for 5 minutes.Tail lysate was diluted either 1:4 (for adult mice) or 1:10 (for embryos or pups) in nuclease-free water, and 1 µl of the diluted lysate was used in a 20 µl PCR reaction.Where indicated, realtime PCR probes were designed to genotype mice or tissues using Transgenic Genotyping Services at JAX and Transnetyx.The list of primers used in this study is available in Supplemental Table 2.

Cochlear Tissue Preparation and Fluorescence Microscopy
The inner ears were extracted from neonatal mice at postnatal day 2, and the cochlear sensory epithelium was isolated and exposed via dissection in PBS.Cochlea were xed for 15 minutes in 4% paraformaldehyde (PFA, 15710, Electron Microscopy Sciences) at room temperature, then permeabilized in PBS with 0.5% Triton X-100.Samples were incubated in 0.5 µM TMR-conjugated HaloTag ligand (G8252, Promega) in PBS for 1 hour at room temperature, then incubated in FITC-conjugated phalloidin (P5282, Sigma) in PBS overnight at 4°C.Samples were washed 4 times in PBS with 0.05% Triton X-100, then post-xed for 1 hour in 4% PFA at room temperature.Whole-mounts were prepared in 10% Mowiol (475904, EMD/Millipore; in 0.1 M tris-Cl, pH 8.5) on positively charged slides (M1021, Denville) with 18 x 18 mm No 1.5 coverslips (48366-045, VWR).Images were collected with Zeiss LSM 800 confocal laser-scanning microscope with a 63x NA1.4 oil immersion objective using the ZEN 2.6 software (Carl Zeiss AG) and enhanced in Adobe Photoshop CC 2023.For measurements in Fig. 3F, recombined cells were identi ed by the enrichment of TMR HaloTag ligand signal at the tips of hair cell cytoskeletal projections.Quantities of recombined and still oxed cells were counted in ImageJ using the Cell Counter plugin.

Histology
Performed as described previously 60 .Brie y, pregnant dams were euthanized by CO 2 asphyxiation.Following CO 2 asphyxiation, embryos were washed in ice-cold phosphate-buffered saline.The embryos (E15.5 to E17.5) were then preserved overnight in 10% neutral buffered formalin before being moved to a 70% ethanol solution.Subsequently, the embryos were para n embedded, sectioned in the mid sagittal plane, and stained with hematoxylin and eosin.

Ethics statement
Overview of the pipeline and the methods used to assess the e ciency of recombination and mosaicism.
(a) In this study, we a thorough characterization pipeline several crucial steps.Our initial focus was on characterizing three unique global Cre-driver strains, achieved by mating female Cre-driver strains with male Rosaoxed strains.The resulting F1 offspring were genotyped using either standard PCR or probe-based assays.The genetic material for these assays was derived from tail tips of E17.5 stage embryos or postnatal P1 to P3 pups.For tissue-speci c analysis of recombination and mosaicism, we conducted histological and immunohistochemical analyses using whole embryos from E15.5 to E17.5 or P2 pups, obtained from the mating of female oxed and male tissue-speci c Cre-strains.(b)The percentage of progeny recombination was determined by dividing the number of litters showing complete recombination by the total number of litters and multiplying it by 100.The percentages of progeny mosaicism and oxed patterns were determined in a similar fashion.oxed allele and a post-cre recombined allele.Note that there is a 10-fold difference in the inter-loxP distance compared to the oxed allele analyzed in (a).(d) Homozygous oxed male mice were mated with either female Ella-cre, CMV-cre, or Sox2-cre mice, and the F1 offspring were genotyped to screen for oxed and recombined alleles.(e) The lengths of all protein-coding genes in mice are arranged in ascending order.Gene lengths varied widely from 0.066 kb to 2270 kb.The median length of protein-coding genes in mice is 15.8 kb.(f)Homozygous oxed male mice with a 10 kb inter-loxP distance were mated with Sox2-cre mice, and the F1 offspring were genotyped to screen for oxed and recombined alleles.Only 22% of the offspring showed complete recombination, while 41% were mosaic, and the remaining 37% showed no recombination.(g) Homozygous oxed male mice with a 12 kb inter-loxP distance were mated with Sox2-cre mice, and the F1 offspring were genotyped.None of the offspring showed complete recombination; however, 14% of the offspring were mosaic.(h) Homozygous oxed male mice with a 15 kb inter-loxPdistance were mated with Sox2-cre mice, and the F1 offspring were genotyped using PCR from tail DNA to screen for oxed and recombined alleles.All the offspring had intact oxed alleles and showed no signs of recombination or mosaicism.
Impact of inter-loxP distances on Cre-mediated recombination and mosaicism in DIO mouse strains: from genotyping to single-cell analysis.
(a) Study design for generating conditional alleles at a xed locus (ROSA26) using the Cas9+Bxb1 toolbox.The Cas9+Bxb1 toolbox allows for accurate and e cient integration of large DNA constructs into a speci c location, achieving higher e ciency compared to Cas9-mediated homology-directed repair (HDR).Bxb1 utilizes attP and attBattachment sites for DNA transgenesis.In our study, we strategically placed an attP attachment site within the ROSA26 safe harbor locus of the B6/J mouse strain using CRISPR/Cas9-mediated HDR.This approach enabled us to seamlessly integrate DNA constructs with DIO alleles ranging from approximately 1 to 8 kb in size.Consequently, we successfully generated conditional knockout mice with single-copy transgenic modi cations.(b, c, and d) Homozygous oxed male mice were mated with either female Ella-cre (b), CMV-cre (c), or Sox2-cre (d) mice, and the F1 offspring were genotyped using PCR from tail DNA to screen for oxed and recombined alleles.Offspring with complete recombination were categorized as recombined, those with no recombination as still oxed, and those with both recombined and oxed alleles as mosaic.The x-axis denotes the inter-loxP distance, and the y-axis denotes the percentage of recombination or mosaicism.The number of offspring genotyped to assess the percent of three possible outcomes: complete recombination, mosaicism, or no recombination are summarized in Supplementary Fig.  Inter-loxP distance and site type in uence on Cre-mediated recombination e ciency and mosaicism.
(a) Male homozygous oxed mouse strains, with a xed inter-loxP of 6.9 kb at different loci and carrying either wildtype loxP sites or mutant loxP sites (lox71 and lox66), were bred with female Ella-cre, CMV-cre, or Sox2-cre mice.(b) Male homozygous oxed mouse strains, with an inter-loxP distance of 2.7 kb and 2.9 kb at different loci, having wildtype loxP sites and mutant lox71/66 sites, respectively, were bred with female Sox2-cre mice.(c) Male homozygous oxed mouse strains, with an inter-loxP distance of 6.9 kb or 2.9 kb at the same locus and with mutant loxP sites (lox71 and lox66), were bred with female Sox2-cre mice.In Figures 5a, b, and c, 6.9 kb, and Rosa-8 kb-were bred with female Ella-cre or Sox2-cre mice.The F1 offspring were then genotyped using PCR and categorized as recombined, still oxed, or mosaic.Note that the percentages were rounded to the nearest whole percent.
Age-dependent recombination e ciency in mouse breeders and its impact on Cre-mediated recombination of oxed alleles.Genome-wide characterization of homozygous conditional knockout mouse strains using Sox2-cre recombination.
Twelve different male homozygous conditional KO strains, with inter-loxP distances ranging from 0.8 to 3 kb at various loci across the mouse genome, crossed with the Sox2-cre strain.The F1 offspring were then genotyped to identify oxed, mosaic, and recombined alleles.Note that the percentages were rounded to the nearest whole percent.
Supplementary Files

Figures
Figures

Figure 3 Cre
Figure 3 3b. (e) Mosaicism at the single-cell level: Confocal image of an 8-kb Myo15a-Halo DIO oxed allele x CMV-cre F1 cochlear sensory epithelium at postnatal day 2. Phalloidin stains lamentous actin (F-Actin), labeling the sensory hair cell cytoskeleton.TMR-conjugated Halo Tagligand labels Halo-Myo15a, the product of recombination.Halo-Myo15aenriches at the tips of hair cell cytoskeletal projections.White asterisks indicate recombined cells expressing Halo-Myo15a.Yellow arrowheads indicate cells that escaped recombination, where the allele is still oxed.(f) Quanti cation of percentages from four different pups as points and the averages by bar plot.

( a )
Representation of mouse breeders ranging age from 5 to 38 weeks was used to determine recombination e ciency.(b) Effects of age on Cre-mediated recombination of four different oxed alleles.Each data point represents the percentage of recombination in a litter of pups, coded by cross of origin, as labelled on plot.X-axis is the age (in weeks) of the Cre-driver breeder at the time of progeny birth.

Table 1
is available in the Supplementary Files section.